Independent insulin signaling modulators govern hot avoidance under different feeding states

Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. Herein, we showed that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. We identified that hot stimulus increases the activity of α′β′ mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, we showed that α′β′ MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α′β′ MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. We observed that Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α′β′ MBn to regulate HAB in different feeding conditions. Finally, we showed that the 2 α′β′-related MB output neurons (MBONs), MBON-α′3 and MBON-β′1, are necessary for the output of integrated hot avoidance information from α′β′ MBn. Our results demonstrate the presence of dual insulin modulation pathways in α′β′ MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.


Introduction
Temperature directly affects several biological processes, such as enzymatic reactions within the body and reproduction.The avoidance of unfavorable environmental temperatures is an innate behavior in all animals, from tiny flatworms to largest fish in the world, the whale sharks [1,2].Thermosensation and temperature avoidance behavior are important for avoiding extremely hot or cold conditions and regulating the body temperature, both of which are critical for survival [3].The fruit fly Drosophila melanogaster has a small size and is highly sensitive to external temperatures.Fruit flies prefer an ambient temperature (approximately 25˚C) and are able to accurately detect a suitable environment for dwelling.Thermosensation in Drosophila relies on multiple classes of thermoreceptors in the last antennal segment of the arista [4][5][6].There are at least 4 classes of thermoreceptors in Drosophila, including receptors for innocuous (harmless) heat, receptors for noxious (harmful) heat, receptors for innocuous cold, and receptors for noxious cold [4][5][6].The hot cell (HC) neurons in the arista of the antenna [6] and the anterior cell (AC) neurons inside the fly brain [5] are majorly function as sensors for harmless hot stimulus.The hot stimulus is conveyed from the antenna lobe to the higher brain center, including the dendritic region of the mushroom bodies (MBs) called the calyx, lateral horn (LH), and posterior lateral protocerebrum (PLP) via the medial antennal lobe tract (mALT) [7].In addition to mALT, the lateral antennal lobe tract (lALT) and transverse 3 antennal lobe tract (t3ALT) can also convey hot stimuli from the antenna lobe to the LH and PLP, but not to the MB [7].
Hunger is an uneasy or painful sensation due to food deprivation.The state of hunger triggers various animal behaviors to meet energy and nutritional requirements.Hunger reduces the core body temperature in mammals [8].Insulin is a peptide hormone secreted by the pancreatic β-cells and plays a major role in energy homeostasis by regulating the blood glucose levels in humans [9].In invertebrates, the evolutionary conserved insulin-like peptides play crucial roles in regulating metabolism, growth, and longevity.Approximately 40 insulin-like peptides have been identified in Caenorhabditis elegans [10].In D. melanogaster, 8 insulin-like peptides (Dilps) and 1 Dilp receptor (InR) have been identified [11].Different Dilps are produced by distinct cell types or tissues during different developmental and adult stages [11].In the adult fly brain, 14 insulin-producing cells (IPCs) are located in the pars intercerebralis (PI) and express Dilp2, Dilp3, and Dilp5, whereas Dilp1 is additionally expressed in larval IPCs [12][13][14][15].Dilp2 secretion is dependent on the nutritional status; moreover, nutrient deprivation inhibits the secretion of Dilp2 by IPCs [16,17].Dilp6 is produced by the adult fat body, and dilp6 mRNA levels are increased during starvation.The secretion of Dilp6 from the fat body is responsible for the starvation-induced reduction in preferred temperature (Tp) [18].In addition, overexpression of dilp6 in the fat body represses the expression of dilp2 and dilp5 mRNA in the brain and reduces the secretion of Dilp2 [19].
MB is a huge brain structure comprising approximately 2,000 MB neurons (MBns), called Kenyon cells in each brain hemisphere, and can be further classified into αβ, γ, and α 0 β 0 MBn according to the distribution of their axons [20].Studies have demonstrated that MBn plays a role in Drosophila temperature preference behaviors via dopamine and cAMP signaling [21][22][23].It has also been shown that MBn integrates satiety and hunger signals to regulate foodseeking behaviors [24,25].However, whether satiety and hunger signals affect thermosensation and temperature preferences in flies, is still unclear.Herein, we showed that hungry flies exhibit a stronger hot avoidance behavior (HAB) compared to food-sated flies.However, hungry and sated flies exhibited no difference in cold avoidance behavior.We showed that hot signals are conveyed by the cholinergic mALT projection neurons, the axons of which are functionally connected to the dendritic region of α 0 β 0 MBn.We also revealed that hot stimulus evokes the same level of calcium response in mALT projection neurons in sated and hungry flies.Our behavioral data suggest that MB activity is required for HAB and α 0 β 0 MBn plays a crucial role in both sated and hungry states.Live brain imaging showed a stronger calcium response to hot stimuli in α 0 β 0 MBn, particularly in the hungry state.Genetic expression of the constitutively active form of InR in α 0 β 0 MBn inhibited the calcium response to hot stimuli and consequently, reduces HAB.In addition, expressing the dominant-negative form of InR in α 0 β 0 MBn caused the opposite effect, suggesting that insulin signaling in α 0 β 0 MBn negatively regulates hot sensation.It has been shown that dilp2 transcript represents approximately 80% of all dilp transcripts present in IPCs [26].RNAi-mediated silencing of dilp2 in IPCs increased HAB and α 0 β 0 MBn activity only in the sated state.Interestingly, dilp6 silencing in the fat body increased HAB and α 0 β 0 MBn activity, specifically in the hungry state.We further showed that PI3K/AKT signaling in α 0 β 0 MBn mediates HAB in sated flies, whereas Ras/ERK signaling in α 0 β 0 MBn mediates HAB in hungry flies.Finally, we identified 2 α 0 β 0 MBn downstream circuits, the MB output neuron (MBON)-α 0 3 and MBON-β 0 1, in which neuronal activity is required for HAB execution.Our results demonstrate that distinct Dilp signals mediate α 0 β 0 MBn activity for proper HAB under different feeding states in Drosophila.

MB activity regulates HAB
D. melanogaster is a small ectotherm whose body temperature is close to the temperature of its surroundings.Drosophila avoid extremely hot and cold environments and choose appropriate surrounding temperature (approximately 25˚C) for habitation [4][5][6].We asked whether the internal feeding state affects the selection of appropriate surrounding temperature in flies.We used a thermoelectric device for our temperature preference behavioral analysis (Figs 1A and S1A-S1C, and S1 Video).To verify the accuracy and stability of the thermoelectric device, we measured the temperature in each quadrant of the plate using temperature sensors for 1 h at 25˚C and at various test temperatures (15˚C, 17˚C, 19˚C, 21˚C, 23˚C, 27˚C, 29˚C, 31˚C, 33˚C, and 35˚C).The margin of error on each aluminum plate was less than 1˚C (approximately 0.5˚C) at all test temperature settings (S1D Fig) .Using this thermoelectric device to perform the two-choice assay [6,7], we found that hungry flies prefer to stay at a surrounding temperature of approximately 23˚C rather than 25˚C (Fig 1B ), which is consistent with the results of a previous study showing that starvation reduces T p in Drosophila [18].Interestingly, we observed an increased hot avoidance by hungry flies compared to food-sated flies; however, no differences were observed in the cold avoidance behavior (Fig 1B).It has been shown that mALT projection neurons convey hot stimuli from the antenna lobe to the calyx of the MB [7], and a recent study also suggests that hunger/satiety signals modulate the MB circuits [25], implying that MB is the integrative center for hot and hunger/satiety signals.Next, we investigated whether silencing or activating the MBn activity affects HAB.Constitutive silencing of the MBn activity by expressing the inward rectifier potassium channel Kir2.1 via VT30559-GAL4 reduced HAB in both hungry and sated states (S2A and S3A Figs).To avoid the effects of constitutive Kir2.1 expression on neuronal development, we used optogenetic tools for temporal silencing or activation of MBn.Temporal silencing of the MBn activity by the blue light gated anion channel GtACR2 [27]    DN and InR CA expression, we co-expressed the tub-GAL80 ts transgene for the acute control of InR DN and InR CA expression.Similar behavioral phenotypes were observed in flies with acute expression of InR DN or InR CA in α 0 β 0 MBn (Figs 2E, 2F, S4F and S5B).Furthermore, acute expression of InR DN or InR CA in α 0 β 0 MBn had <0.0001, 0.0002, <0.0001, and 0.001 from left to right; Hunger: P-values: 0.4572, 0.3521, 0.8352, and 0.855 from left to right).(F) Optogenetic silencing of αβ MBn activity inhibited HAB in the sated state but not in the hungry state (Satiety: P-values: < 0.0001, 0.0003, 0.0002, and 0.0019 from left to right; Hunger: P-values: 0.3912, 0.7685, 0.8361, and 0.4902 from left to right).(G) Optogenetic silencing of α 0 β 0 MBn activity inhibits HAB in both states (Satiety: P-values: <0.0001, 0.0034, <0.0001, and 0.0019 from left to right; Hunger: P-values: <0.0001, 0.0027, <0.0001, and 0.0002 from left to right).(H) Hot stimuli induced the calcium response in γ MBn during satiety, while during hunger, the hot response was diminished (P = 0.0002).The GCaMP intensity changes (ΔF/F 0 ) in MB γ lobe were recorded and analyzed.(I) Hot stimuli induced the calcium response in αβ MBn in the sated state, while in hungry state, this hot response was diminished (P = 0.005).The GCaMP intensity changes (ΔF/F 0 ) in MB β lobe were recorded and analyzed.(J) Hot stimuli induced the calcium response in α 0 β 0 MBn during satiety, whereas this hot response was enhanced during hunger (P = 0.0373).The GCaMP intensity changes (ΔF/F 0 ) in MB β 0 lobe were recorded and analyzed.The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in behavioral assays (B-G) or a single fly in live brain calcium imaging experiments (H-J).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (C-G) or the unpaired two-tailed t test (B, H-J).*P < 0.05.HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.https://doi.org/10.1371/journal.pbio.3002332.g001no effect on the cold avoidance behavior, suggesting that Dilp signaling in MB mediates hot but not cold responses (Figs 2G, 2H, S4G, S4H and S5C).Live brain imaging data showed that InR DN transgene expression enhanced the hot stimuli-induced calcium response, whereas InR CA expression reduced the calcium response in α 0 β 0 MBn in both sated (Fig 2I ) and hungry (Fig 2J ) flies.Together, our data suggest that InR negatively regulates α 0 β 0 MBn activity, which contributes to HAB in both sated and hungry states.
Dilp2 mediates α 0 β 0 MBn activity required for HAB during satiety via PI3K/AKT signaling In the fly brain, Dilp2, Dilp3, and Dilp5 are released from IPCs during satiety; therefore, we investigated HAB in dilp mutant flies.Behavioral screening showed significantly increased HAB in sated dilp2 but not dilp3 and dilp5 mutant flies (Figs 3A and S6A).Since α 0 β 0 MBn is critical for the hot response, we investigated whether loss of Dilp2 affects α 0 β 0 MBn response to the hot stimulus.Live brain imaging data showed an increased calcium response to the hot stimulus in α 0 β 0 MBn of dilp2 mutant flies in the sated state (Fig 3B, left panel).However, the increased calcium response in α 0 β 0 MBn was not observed in hungry dilp2 mutant flies, further suggesting that Dilp2 mediates hot responses only during satiety (Fig 3B, right panel).Since Dilp2 is secreted by IPCs in the brain, we investigated HAB in IPCs-specific dilp2 knockdown flies (S6B Fig) .To exclude the developmental effect of decrease IPCs-specific dilp2 expression, we co-expressed the tub-GAL80 ts transgene for adult-stage-specific silencing of dilp2 in IPCs increased HAB in the sated but not in the hungry state (Figs 3C and S6C), suggesting that Dilp2 secreted by IPCs inhibits HAB only during satiety.Our immunohistochemistry data also showed abundant accumulation of Dilp2 in IPCs during the hungry state (S6D Fig), which is consistent with the results of previous studies in Drosophila larvae [16] and adult flies [17].We performed live brain imaging to determine whether the Dilp2 secretion by IPCs represent satiety or a hot signal.Live brain imaging data showed that IPCs are not responsive to hot stimuli, which indicates that Dilp2 secretion represents the sated state rather than the hot signal (S6E Fig).
To assess whether Dilp2 produced by IPCs inhibits the hot response of α 0 β 0 MBn, we genetically silenced dilp2 in IPCs and recorded the calcium response in α 0 β 0 MBn before and after the hot stimulus.Results showed that silencing dilp2 in IPCs significantly increased the calcium response to hot stimulus in α 0 β 0 MBn in sated but not in hungry flies (Fig 3D).Furthermore, dilp2 overexpression in IPCs inhibited HAB (Figs 3E and S6F), and live brain imaging P-values: <0.0001, 0.0035, <0.0001, and 0.485 from left to right; Hunger: P-values: <0.0001, <0.0001, 0.0005, and 0.0022 from left to right).(C) Genetic expression of InR DN in α 0 β 0 MBn increased HAB in both sated and hungry states (Satiety: P-values: <0.0001, <0.0001, <0.0001, and 0.0506 from left to right; Hunger: P-values: 0.0016, <0.0001, 0.0309, and 0.0562 from left to right).(D) Genetic expression of InR CA in α 0 β 0 MBn decreased HAB in both sated and hungry states (Satiety: P-values: <0.0001, 0.0002, 0.0004, and 0.0138 from left to right; Hunger: P-values: 0.0003, 0.0043, 0.0003, and 0.4695 from left to right).(E) Adult-stage-specific expression of InR DN in α 0 β 0 MBn increased HAB in both sated and hungry states (Satiety: P-values: <0.0001, 0.0012, <0.0001, and 0.2074 from left to right; Hunger: P-values: <0.0001, 0.0002, 0.0418, and 0.0035 from left to right).(F) Adult-stage-specific expression of InR CA in α 0 β 0 MBn decreased HAB in both sated and hungry states (Satiety: P-values: 0.0013, 0.0379, 0.008, and 0.0198 from left to right; Hunger: P-values: 0.0094, 0.0051, 0.0246, and 0.0155 from left to right).(G) Adult-stage-specific expression of InR DN in α 0 β 0 MBn did not affect cold avoidance behavior in both sated and hungry states (Satiety: P-values: 0.7397, 0.9641, 0.8158, and 0.7703 from left to right; Hunger: P-values: 0.803, 0.7378, 0.9292, and 0.8924 from left to right).(H) Adult-stagespecific expression of InR CA in α 0 β 0 MBn did not affect cold avoidance behavior in both sated and hungry states (Satiety: P-values: 0.908, 0.7472, 0.8053, and 0.6225 from left to right; Hunger: P-values: 0.8746, 0.8515, 0.9402, and 0.2481 from left to right).(I, J) Genetic expression of InR CA decreased hot-induced calcium response, whereas expression of InR DN increased hot-induced calcium response in α 0 β 0 MBn compared to control groups in sated (I) and hungry (J) flies (P = 0.0145 and 0.0102 for satiety; P = 0.0004 and 0.0168 for hunger).The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.The GCaMP intensity changes (ΔF/F 0 ) in MB β 0 lobe were recorded and analyzed in each calcium imaging data.Each N represents either a group of 15 flies analyzed together in the behavioral assay (A-H) or a single fly in calcium imaging experiments (I, J).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (A-H) or the unpaired two-tailed t test (I, J). *P < 0.05.HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.
Blocking the PI3K/AKT signaling in α 0 β 0 MBn increased HAB in sated flies; however, it had no effect on HAB in hungry flies (Figs 3G and S7A-S7G).There are 2 major cellular signaling pathways, PI3K/AKT and Ras/ERK pathways, which can be activated by InR [29][30][31].We asked whether other Dilp-InR signals regulate HAB and α 0 β 0 MBn activity during the hungry state.Intriguingly, RNAi-mediated knockdown of Ras, Raf, or Erk in α 0 β 0 MBn induced a strong HAB only in the hungry state but not in the sated state (S9A-S9F Considering that the phosphorylation of AKT and ERK represents the activity of PI3K/ AKT and Ras/ERK signaling pathways [30,32], we asked whether the expression of phospho-AKT (pAKT) and phospho-ERK (pERK) in α 0 β 0 MBn in sated flies differs from that in hungry flies.Immunohistochemical analysis revealed higher expression levels of pAKT in the sated state (Fig 5A ), whereas higher expression levels of pERK in hungry flies were observed (Fig 5B).However, the genetic expression of InR DN in α 0 β 0 MBn ameliorated the increased levels of pAKT and pERK in sated and hungry flies, respectively (Fig 5C and 5D).The genetic silencing of dilp2 in IPCs also suppressed the levels of pAKT in sated flies (Fig 5E), whereas silencing of dilp6 in the fat body suppressed the levels of pERK in hungry flies (Fig 5F).Overall, our results suggest that satiety-induced Dilp2 secretion by IPCs triggers an intracellular signaling in α 0 β 0 MBn, which is distinct from hunger-induced Dilp6 secretion from the fat body.These 2 distinct InR-dependent intracellular signals contribute to proper HAB under both feeding states in Drosophila.(A) dilp6 loss-of-function mutant flies (dilp6 LOF ) showed increased HAB when hungry (Satiety: P-values: 0.6176, 0.4785, 0.8634, and 0.7292 from left to right; Hunger: P-values: 0.0003, 0.0029, 0.0222, and 0.4018 from left to right).(B) α 0 β 0 MBn showed increased hot response in dilp6 LOF mutant background specifically when hungry but not when sated (P = 0.2043 for satiety; P = 0.0002 for hunger).(C) Adult-stage-specific knockdown of dilp6 in the fat body increased HAB in hungry but not in sated flies (Satiety: Pvalues: 0.7112, 0.4332, 0.748, and 0.4753 from left to right; Hunger: P-values: 0.0006, 0.0002, 0.0332, and 0.0732 from left to right).(D) Genetic knockdown of dilp6 in the fat body increased hot response of α 0 β 0 MBn specifically in the hungry state (P = 0.8682 for satiety; P = 0.0328 for hunger).(E) Adult-stage-specific expression of dilp6 in the fat body decreased HAB in the hungry state (Satiety: P-values: 0.2437, 0.2066, 0.3148, and 0.2978 from left to right; Hunger: P-values: <0.0001, <0.0001, <0.0001, and 0.0194 from left to right).(F) Genetic expression of dilp6 in the fat body decreased hot response of α 0 β 0 MBn in the hungry state (P = 0.5214 for satiety; P = 0.002 for hunger).(G) Adult-stage-specific knockdown of Erk in α 0 β 0 MBn increased HAB during hunger (Satiety: P-values: 0.9053, 0.6805, 0.4785, and 0.2593 from left to right; Hunger: P-values: <0.0001, 0.0011, 0.0026, and 0.108 from left to right).(H) Genetic knockdown of Erk in α 0 β 0 MBn increased hot responses of α 0 β 0 MBn during hunger but not during satiety (P = 0.2155 for satiety; P = 0.0361 for hunger).(I) Adult-stage-specific expression of Erk in α 0 β 0 MBn decreased HAB during the hungry state (Satiety: P-values: 0.6868, 0.7727, 0.7495, and 0.0552 from left to right; Hunger: P-values: 0.001, 0.0003, 0.0002, and 0.0121 from left to right).(J) Genetic expression of Erk in α 0 β 0 MBn decreased the hot response of α 0 β 0 MBn in the hungry state (P = 0.4394 for satiety; P = 0.0003 for hunger).The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in the behavioral assay (A, C,  E, G, I A, B, D, F, H, J). *P < 0.05; ns, not significant.HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.
https://doi.org/10.1371/journal.pbio.3002332.g004mALT conveys the hot signal to α 0 β 0 MBn via the cholinergic transmission The mALT projection neurons convey thermal stimuli from the antenna lobe to the MB, LH, and PLP regions in the fly brain [7].We investigated whether α 0 β 0 MBn responses to hot stimuli are delivered via mALT and the neurotransmitter involved in this process.To address this, we labeled the mALT neurons using VT40053-GAL4 (Fig 6A) [7].The axons of these neurons innervate the dendritic region of the MB, also known as calyx (Fig 6B).Immunostaining using the anti-choline acetyltransferase (anti-ChAT) antibody showed positive signals in mALT axons, suggesting that these neurons use acetylcholine for neurotransmission (Figs 6C and S10A) [33].Furthermore, kir2.1 transgene expression in mALT neurons reduced HAB in both sated and hungry states, suggesting that mALT neuronal activity is required for normal HAB (S10B Fig) .To avoid the developmental effects of kir2.1 transgene expression in neurons, Next, we asked whether α 0 β 0 MBn shows similar hot responses in hunger and sated states when the InR signal is suppressed.We genetically expressed InR DN in α 0 β 0 MBn via VT30604-GAL4 and performed live calcium brain imaging.Results showed that calcium responses to hot stimuli did not significantly differ between both feeding states (Fig 6H).Moreover, we also investigated the α 0 β 0 MBn response to hot stimuli in dilp2 -/-and dilp6 LOF double mutant flies.We found that the calcium response to hot stimuli did not significantly differ during both feeding states (Fig 6I).Overall, these results indicate that cholinergic mALT neurons are the major input for hot stimuli in α 0 β 0 MBn in both sated as well as hungry flies and that Dilp2 and Dilp6 are only involved in the suppression of α 0 β 0 MBn activity under sated and hungry states, respectively (Figs 2-6).

MBON-α 0 3 and MBON-β 0 1 are essential for HAB execution
Since the hot input is conveyed by cholinergic mALT to α 0 β 0 MBn (Fig 6), whereas Dilp2 and Dilp6 regulate α 0 β 0 MBn activity during sated and hungry states, respectively (Figs 3-5), we investigated the output neurons that are critical for HAB.The information present in the MB can be read out by specific MBONs, the dendrites of which are restricted to different domains  <0.0001, 0.0001, and 0.0271 from left to right; Hunger: P-values: 0.0001, 0.001, 0.0103, and 0.001 from left to right).(E) Adult-stage-specific knockdown of ChAT in mALT inhibited HAB during both feeding states (Satiety: P-values: <0.0001, <0.0001, 0.0029, and <0.0001 from left to right; Hunger: P-values: <0.0001, 0.0005, <0.001, and <0.0001 from left to right).(F) Hot stimulus evoked the calcium response in hot glomerulus of mALT and this calcium response was not significantly different between sated (black) and hungry (red) flies (P = 0.7554).The GCaMP intensity changes (ΔF/F 0 ) in mALT dendrites were recorded and analyzed.(G) Optogenetic activation of mALT via CsChrimson evoked calcium responses in α 0 β 0 MBn in flies carry R35B12-lexA/CsChrimson; VT40053/lexAop-GCaMP7 (green) but not in flies carrying R35B12-lexA/CsChrimson; +/lexAop-GCaMP7 flies (black) (P < 0.0001 for satiety; P < 0.0001 for hunger).(H) The calcium responses to hot stimuli in α 0 β 0 MBn do not differ significantly between sated (black) and hungry (red) flies when InR DN transgene is expressed in α 0 β 0 MBn (P = 0.6093).(I) The calcium responses to hot stimuli in α 0 β 0 MBn do not differ significantly between sated (black) and hungry (red) dilp2 -/-and dilp6 LOF double mutant flies (P = 0.4850).The GCaMP intensity changes (ΔF/F 0 ) in MB β 0 lobe were recorded and analyzed.The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in the behavioral assay (D, E) or a single fly in calcium imaging experiments (F-I).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (D, E) or the unpaired two-tailed t test (F-I).*P < 0.05; ns, not significant.HAB, hot avoidance behavior; mALT, medial antennal lobe tract; MBn, mushroom body neuron; SEM, standard error of mean.https://doi.org/10.1371/journal.pbio.3002332.g006 of MB lobes [34].It has been reported that the postsynaptic region of the MBON has high plasticity, and different MBONs encode distinct intrinsic valences, which can drive the approach or avoidance behavior in Drosophila.Therefore, summarizing valences in MBONs might be important for achieving a suitable behavioral output [34,35].There are at least 9 different types of MBONs whose dendrites are restricted to distinct subdomains of α 0 β 0 lobes [34,36] (S3H-S3P Fig) .We individually tested the involvement of these α 0 β 0 -related MBONs in regulating HAB by GtACR-mediated silencing of neuronal activity (Figs 7A, 7B and S11).Our results showed that transient inhibition of MBON-α 0 3 (Fig 7A ) or MBON-β 0 1 activity (Fig 7B ) during behavioral assays disrupted HAB in both sated and hungry states.In addition, live brain imaging also indicated that MBON-α 0 3 and MBON-β 0 1 respond to hot stimuli in both feeding states (Fig 7C and 7D).These results suggest that the integrated information from α 0 β 0 MBn is transmitted through MBON-α 0 3 and MBON-β 0 1 for proper HAB execution by sated and hungry flies.

Discussion
In mice, it has been reported that insulin directly inhibits the activity of warm-sensitive neurons in the hypothalamus suggesting that insulin suppresses the sensation of hot stimuli [37].The effects of insulin signaling on the physiological response to heat stress are now being explored.In this study, we revealed the cellular mechanisms underlying the regulation of insulin signaling for sensing hot temperatures under different feeding conditions in Drosophila.Flies prefer to stay at relatively higher temperatures in the food-sated state (Fig 1B).This differential response to the hot stimulus is tightly controlled by distinct insulin signals that produce .We demonstrated that flies exhibit a stronger HAB in the hungry state than in the sated state.Our data suggest that in the sated state, Dilp2 secreted by IPCs is responsible for conveying the satiety information to α 0 β 0 MBn, which inhibits α 0 β 0 MBn activity by inducing the PI3K/AKT signaling (Fig 3).In contrast, under hungry conditions, Dilp6 is released from the fat body to convey the hunger signal to α 0 β 0 MBn, which inhibits α 0 β 0 MBn activity by inducing the Ras/ERK signaling (Fig 4).Our immunohistochemistry data also support the notion that increased levels of pAKT and pERK in α 0 β 0 MBn under sated and hungry states, respectively (Fig 5).Our results further suggest that Dilp2 increases pAKT levels while Dilp6 increases pERK levels in α 0 β 0 MBn during sated and hungry states (Fig 5E and 5F).However, we cannot totally rule out the possibility that Dilp2 also increases pERK levels and Dilp6 also increases pAKT levels in α 0 β 0 MBn.Here, we propose that different intracellular signals induced by Dilp2 and Dilp6 under different feeding states, both of which suppress the hot response of α 0 β 0 MBn (Fig 8).The hot stimulus is conveyed through cholinergic mALT neurons to α 0 β 0 MBn (green) and α 0 β 0 MBn activity is positively correlated with HAB.In the food-sated state, Dilp2 is secreted from IPCs to inhibit the hot response of α 0 β 0 MBn by inducing PI3K/AKT signaling that causes a weak HAB and consequently flies decrease their hot avoidance for increasing their metabolism.On the other hand, Dilp2 is no longer released from IPCs in the hungry state.Instead, Dilp6 is released from the fat body to inhibit the hot response of α 0 β 0 MBn by inducing Ras/ERK signaling.The inhibition efficiency of Ras/ERK in the hungry state is not as strong as PI3K/AKT signaling, consequently flies exhibit strong hot responses in α 0 β 0 MBn that contributes to a strong HAB, thereby increasing the hot avoidance of flies to reduce their metabolism for saving energy in the hungry state.HAB, hot avoidance behavior; IPC, insulin-producing cell; mALT, medial antennal lobe tract; MBn, mushroom body neuron; SEM, standard error of mean.https://doi.org/10.1371/journal.pbio.3002332.g008 A previous study showed that overexpression of Dilp6 in adult fat body represses Dilp2 and Dilp5 expression in IPCs and the secretion of Dilp2 into the hemolymph [19].Our anti-Dilp2 immunostaining data also showed that genetic knockdown of dilp6 in the fat body increased the Dilp2 expression in IPCs.In contrast, overexpression of dilp6 in fat body decreased Dilp2 expression in IPCs, in both sated and hungry states (S12 Fig) .It has been shown that Dilp6 expression in the fat body reduces short neuropeptide F (sNPF) expression in the brain.This sNPF reduction may correlate to the Dilp2 suppression in IPCs [19,38].Reducing circulating Dilp2 could contribute to lifespan extension and the correlated changes in carbohydrate/lipids storage and oxidative stress resistance.We postulate that Dilp6 secretion in fat body during starvation not only suppresses the Dilp2 expression in IPCs but also induces the Ras/ERK signaling in α 0 β 0 MBn that contributes to the hungry state-dependent HAB (Fig 4).
In contrast to Dilp2, Dilp3, and Dilp5, it has been shown that Dilp1 is transiently expressed in IPCs, specifically during pupa and early stage of adult flies [39,40].The immunohistochemistry data of Dilp1 showed that Dilp1 is expressed in newly eclosed adult flies and dramatically decreases after 1 week [39].However, the Dilp1 expression in IPCs of adult flies lasts over 6 weeks and only slightly decreases after 7 weeks in diapause conditions [39,41].It has been proposed that the transient Dilp1 expression during the early stage of adult flies could be associated with a metabolic transition from pupa to adult during normal conditions.Mutations of dilp1 reduce the organism's weight during pupal development, whereas overexpression of dilp1 increases it [40].Survival during starvation decreases significantly in newly eclosed, but not in 6-to 7-day-old dilp1 mutant female flies.In addition, starvation resistance is not affected in dilp1 mutant male flies [40].Therefore, it is unlikely that Dilp1 regulates α 0 β 0 MBn neuronal activity for HAB since we used the 7-to 10-day-old adult flies for experiments in the current study.
A previous study showed that Dilp6 regulates AC neurons activity during starvation [18], which leads to starvation-induced reduction of T p as hungry flies prefer to stay at 23˚C rather than 25˚C, which is consistent with our results (Fig 1B).Manipulation of InR expression in α 0 β 0 MBn had no effect on cold avoidance and T p , suggesting that this signaling is only involved in HAB (Figs 2G, 2H, S4G and S4H).Starvation-induced Dilp6 secretion from the fat body regulates both AC neurons and α 0 β 0 MBn, which contributes to changes in T p and HAB during the hungry state.Although 8 different Dilps are expressed in D. melanogaster, only single InR is present in flies, suggesting that Dilp2 and Dilp6 bind to the same InR in α 0 β 0 MBn, but trigger distinct signaling pathways under different feeding conditions, contributing to different levels of the hot response and HAB.A prior study showed that Dilp2 and Dilp5 differentially modulate the signal transduction kinetics of pAKT in Drosophila S2 cells, by which Dilp2 induces acute and transient phosphorylation of AKT, whereas Dilp5 induces sustained AKT phosphorylation [42].These results support the notion that distinct Dilp ligands could trigger different intracellular transduction kinetics through 1 InR.However, it remains unclear how Dilps binding to the same InR in α 0 β 0 MBn induces distinct intracellular signaling under different feeding states.
Our results revealed that insulin receptors in αβ and γ MBn are not involved in HAB (S4B-S4E Fig) .However, temporal silencing of the αβ or γ MBn activity reduced the HAB in the sated state (Fig 1E and 1F) suggesting that αβ and γ MBn regulate HAB via other mechanisms.Dopamine is an essential neurotransmitter produced by the central and peripheral nervous systems and plays an important role in neuromodulation [43].Several studies have shown that dopamine is involved in thermoregulation.The dopaminergic pathways in the hypothalamus help in improving the tolerance to a high core temperature and slow down the rate of core temperature rise in the human body [44].In rats, dopamine breakdown processes in the preoptic area and anterior hypothalamus are active and play a role in thermoregulation during exercise [45].In flies, the axons of 2 types of dopaminergic neurons, including the protocerebral posterior lateral (PPL) cluster and protocerebral anterior medial (PAM) cluster, innervate the MB lobes, transmitting cold signals to MBn [23,46].It has been shown that PPL1-α3/α 0 3, PPL1-α2α 0 2, PPL1-γ2α 0 1, PPL1-γ1pedc, PAM-β2, and PAM-β 0 2 respond to cold stimuli suggesting their physiological role in conveying cold signals to MB lobes [23,46,47].
Besides temperature sensation, dopaminergic neurons also regulate the Drosophila feeding state and MBn activity.A previous study showed that dopaminergic neurons encode odor/ taste valence and regulate internal physiology in specific MB lobe compartments [48].Other studies have demonstrated that the dopaminergic PPL1-α 0 2α2 neurons only receive satiety signals, whereas PAM-β2β 0 2a and PPL1-γ2α 0 1 neurons receive both satiety and hunger signals [25,49].Additionally, nutritious sugar feeding immediately suppresses the activity of dopaminergic PAM-γ3 neurons, providing a positive reinforcing signal for sugar-reward memory formation [50].Together, the activity of αβ and γ MBn involved in HAB may be regulated by dopaminergic modulations during the sated state in Drosophila.
In Drosophila, hot sensation relies on thermoreceptors in the last antennal segment, the arista [4][5][6].The hot signal is conveyed from the antenna lobe to the higher brain center, including the MB calyx, LH, and PLP, via hot responsive mALT projection neurons [7] (Fig 6A).Our brain imaging data showed co-localized signals in mALT axons and MB dendrites, suggesting synaptic connections between mALT and MBn (Fig 6B).In addition, immunohistochemistry data also suggest that mALT is a cholinergic neuron that transmits acetylcholine to MBn (Figs 6C and S10A).Silencing mALT neuronal activity or ChAT knockdown in mALT reduced HAB in sated and hungry flies, suggesting that mALT conveys hot signals in both feeding states via the cholinergic transmission (Fig 6D and 6E).Optogenetic activation of mALT via CsChrimson induced a significant calcium response in α 0 β 0 MBn, whereas silencing mALT activity abolished the hot response in α 0 β 0 MBn in both feeding states (Figs 6G and S10E).These results indicate that α 0 β 0 MBn is downstream of mALT neurons and receives the hot input from mALT.Interestingly, our live calcium brain imaging data revealed the same hot response level in mALT neurons under both feeding states, indicating that satiety and hunger signals had no effect on the hot response in mALT (Fig 6F).Insulin signaling only suppressed the activity of α 0 β 0 MBn, but not the upstream mALT during exposure to the hot stimulus (Figs 2-6).
Previous studies have demonstrated compartment-specific changes in synaptic strength at MBn-MBON synapses following MBn activation paired with artificial activation of reinforcing dopaminergic neurons, suggesting that the integrated information from MB is transferred to specific MBONs [34,35,51,52].At least 9 different α 0 β 0 -related MBONs have been reported in the fly brain, and some of them play important roles in olfactory memory, visual memory, and sleep regulation in Drosophila [34,36,[53][54][55].Herein, we individually tested the involvement of each α 0 β 0 -related MBON in HAB and revealed that the activity of MBON-α 0 3 and MBON-β 0 1 is required for HAB under sated and hungry states (Fig 7A and 7B).Since the activity of α 0 β 0 MBn was increased in response to the hot stimulus in hungry flies (Fig 1J), these flies also showed an increased calcium response to the hot stimulus in MBON-α 0 3 and MBON-β 0 1 (Fig 7C and 7D).It has been shown that MBON-α 0 3 forms a suppressed short-lived memory trace following aversive olfactory conditioning, whose activity is required for the 15-min memory [56].In addition, MBON-β 0 1 activity is critical for polyamine odor preference and mating behaviors in female flies [57].Our results showed the physiological roles of MBON-α 0 3 and MBON-β 0 1, which are responsive to hot stimuli and regulate HAB execution in Drosophila ( Fig 7).
The reasons why food-sated flies prefer to stay at relatively higher temperatures compared to hungry flies remain unknown.One hypothesis is that under starvation conditions, flies try to save their energy by changing their behaviors and becoming more sensitive to hot stimuli.In contrast, after feeding, flies become less sensitive to hot stimuli and exhibit decreased HAB to approach a relatively high temperature for increasing their metabolic rates.The detailed mechanisms of how nutritional status affects temperature-sensing behaviors in Drosophila remain unknown and would be an interesting topic for future studies.

Temperature avoidance behavior assay
The device used for analyzing temperature avoidance behavior was prepared as described previously [6,8].Each quadrant of the device contained 4 components: a thermoelectric cooling chip with an aluminum plate (6 cm × 6 cm), a temperature sensor, a heat spreader, and a microcontroller.In first and third quadrants, the temperature was set to 25˚C, while the second and fourth quadrants were set to the experimental temperature (15 to 35˚C).The pulsewidth modulation (PWM) is used in the thermoelectric device to control the operating voltage of the thermoelectric cooling module, which decides whether to turn on the power supply of cooling fans of heat spreaders according to different temperature ranges.The microcontroller sets different PWM parameters in different temperature ranges according to the characteristics of the thermoelectric cooling module.The microcontroller transmits information of the aluminum sheets' temperature values and the cooling fans' power status to a computer to monitor the real-time temperature control status.During each trial, the apparatus was covered with glass coated with RainX to prevent flies from escaping the device, and the avoidance index was calculated for each test temperature.The assays were carried out in a room maintained at 24˚C, 50% to 60% humidity.Groups of 15 flies of 7-to 10-day-old age of both sexes were subdued with 98% CO 2 and randomly placed in the arena.The flies were free to move on the aluminum plate, and the movement of flies was recorded for 3 min.The avoidance index was defined as (number of flies at 25˚C-number of flies at the test temperature)/total number of flies.The avoidance indices were compared using the unpaired t test (2 groups) or analysis of variance (ANOVA) (3 groups).
For adult-stage-specific gene knockdown or adult-stage-specific gene expression experiments, the tub-GAL80 ts transgene was introduced for temporal inhibition of GAL4 expression.The experimental group flies were kept at 18˚C throughout development.After eclosion, the flies were shifted to 30˚C for 5 days and shifted back to 24˚C for 12 h before behavioral assays.Our heat shock protocol did not change the HAB compared to that of the flies kept at 24˚C (S13 Fig) .The control group flies were maintained at 18˚C throughout development.After eclosion, flies were shifted to 24˚C for 5.5 days before performing the behavioral assays.All behavioral assays were performed at 24˚C.
For GtACR2-mediated neuronal silencing experiments, temperature avoidance behaviors were recorded under blue light (468 nm) irradiation with an intensity of approximately 2.4 mW/cm 2 for 3 min.For CsChrimson-mediated neuronal activation experiments, temperature avoidance behaviors were recorded under red light (625 nm) irradiation with an intensity of approximately 11.2 mW/cm 2 for 3 min.Flies were received 3 min of blue or red light irradiation during the experiments.

Immunohistochemistry and confocal imaging
Fly brains were dissected in PBS and fixed in 4% paraformaldehyde on ice with 3 repetitions of microwave irradiation (2,450 MHz; 1,100 watts) for 60 s with continuous rotation.Brain samples were then incubated in blocking buffer (PBS containing 10% normal goat serum and 2% Triton X-100) and degassed in a vacuum chamber (depressurized to 270 mmHg then held for 10 min) for 6 cycles.Next, the brains were blocked and permeabilized in blocking buffer at 25˚C for 2 h.The fly brains were immunostained with the mouse 4F3 anti-discs large (DLG) monoclonal antibody (1:10; AB-528203, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-ChAT (1:200; ChAT4B1, Developmental Studies Hybridoma Bank, University of Iowa), rabbit anti-Dilp2 (1:200; Takashi Nishimura's lab), rabbit anti-pAKT (1:200; #9271, Cell Signaling Technology), or rabbit anti-pERK (1:200; #4376, Cell Signaling Technology).Brain samples were incubated in dilution buffer (PBS containing 1% normal goat serum and 0.25% Triton X-100) with the primary antibody at 25˚C for 24 h.After 3 intensive washes in PBS-T, the samples were incubated with biotinylated goat anti-mouse IgG (1:200; B-2763, Thermo Fisher Scientific) or goat anti-rabbit IgG (1:200; B-2770, Thermo Fisher Scientific) at 25˚C for 24 h.Next, the samples were washed and incubated with Alexa Fluor 633 streptavidin (1:500; Invitrogen) at 25˚C for another 24 h.After washing, the samples were cleared and mounted using FocusClear (FC-101, CelExplorer).The samples were covered with coverslips and imaged using the Zeiss LSM 700 confocal microscope with either a 63× glycerin-immersion objective (N.A. = 1.4 and 170 μm working distance) or a 40× C-Apochromat water-immersion objective (N. A. = 1.2 and 220 μm working distance).The pinhole (optical section) was set at 1.5 μm when imaging with the 63× objective lens and at 2 μm for images taken with the 40× objective lens.All images were processed using the ZEN or ImageJ software.

Brain calcium imaging
Live brain calcium imaging experiments were performed as described in our previous study [23].To monitor the changes in intracellular calcium ions in response to the hot stimulus within the fly brain flies expressing GCaMP7s were immobilized using a 250 μl pipette tip.Using fine tweezers, a small opening was made on the head capsule, and a drop of adult hemolymph-like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 8.2 mM MgCl 2 , 4 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 5 mM trehalose, 10 mM sucrose, and 5 mM HEPES (pH 7.5), 265 mOsm) was added immediately to prevent dehydration of the brain.The pipette tip along with the fly was mounted in a perfusion chamber containing 400 μl AHL solution at 24˚C.The hot stimulus was administered by adding an additional 200 μl AHL solution at 55˚C.The final temperature of the solution (approximately 31˚C) was monitored using a thermometer.Timelapse recording of the GCaMP intensity before and after the hot stimulus was performed using the Zeiss LSM700 microscope with a 40× Achroplan IR lens.The 488 nm excitation laser and a detector for emissions passing through a 555 nm short-pass filter were used for time-lapse recording.An optical slice with a resolution of 512 × 512 pixels was continuously monitored for 75 s at a rate of 2 frames per second.For optogenetic experiments, flies were kept in a medium containing 400 mM all-trans-retinal (R2500, SIGMA) for 4 days before performing the calcium imaging assay, as described above.A 625 nm LED was used as the light source to activate CsChrimson-expressing neurons, and GCaMP signals were recorded using the Zeiss LSM700 microscope.The regions of interest (ROIs) were manually assigned to anatomically different neuropils or soma regions and are described in detail in each figure legend.F 0 was defined as the average of 20 frames of fluorescence intensity before the temperature stimulus.ΔF was defined as fluorescence intensity changes after temperature stimulus, which is fluorescence intensity post stimulation minus F 0 .The temperature-induced intensity changes were calculated as ΔF/F 0 , and intensity maps were generated using the ImageJ software; the maximum (max) intensity (8-bit binary digit, 255) is presented by red color, and minimum intensity (8-bit binary digit, 0) is presented by blue color.

Statistical analysis
Raw data were analyzed parametrically using the Prism 6.0 software (GraphPad).Raw data from 2 groups were evaluated using the unpaired two-tailed t test.Raw data from 3 groups were evaluated using one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests.A P-value <0.05 was considered statistically significant.The N values for each experiment are indicated in the figures.All data are presented as mean ± standard error of mean (SEM).

S1 Fig. The introduction of thermoelectric device. (A, B)
The top view (A) and side view (B) of a thermoelectric device.The thermoelectric device contains 6 components: microcontroller system, temperature sensors, aluminum plates, cooling chips, heat spreaders, and a glass cover.The pulse-width modulation (PWM) is used to control the operating voltage of thermoelectric cooling module.The thermoelectric cooling module is used to control the temperature of aluminum plate that range from 15˚C to 35˚C.We set a 6 × 6 cm aluminum sheet on each thermoelectric cooling module to increase conduction velocity.For heat dissipation, we set a heat spreader with aluminum below each cooling chip.Four thermoelectric cooling modules are lined up to form an arena and temperature sensor is added in each thermoelectric cooling module.(C) The distributions of flies on the thermoelectric device during a two-choice assay experiment.(D) The accuracy and stability of the thermoelectric device has been shown.The 2' (light-red region) and 4' (dark-red region) quadrants were individually set to different test temperatures (15, 17, 19, 21, 23, 27, 29, 31, 33, or 35˚C).The 1' (dark-green region) and 3' (light-green region) quadrants were set to 25˚C.The temperatures on each of the 4 quadrants plates were recorded for 3,600 s.The margin of error on each aluminum plate was lower than 1˚C (approximately 0.5˚C) in all test temperature settings.(E, F) Three min of blue or red light irradiations did not alter the setting temperatures of the aluminum plates of the thermoelectric device.The 2' (light-red region) and 4' (dark-red region) quadrants were set to different test temperatures (27, 29, 31, or 33˚C).The 1' (dark-green region) and 3' (light-green region) quadrants were set to 25˚C.The temperature was recorded for 540 s on each aluminum plate, and light irradiations were added from 180 to 360 s.The margin of error on each aluminum plate was lower than 1˚C in blue light (E) and red light (F) irradiation conditions.The data underlying this figure can be found in S1 Data.(TIF)

Fig 2 .
Fig 2. Insulin signaling in α 0 β 0 MBn is critical for HAB.(A) Genetic expression of the dominant negative form of InR (InR DN ) in MBn increased HAB in both sated and hungry states (Satiety: P-values: <0.0001, <0.0001, <0.0001, and 0.0055 from left to right; Hunger: P-values: <0.0001, <0.0001, 0.0026, and 0.0291 from left to right).(B) Genetic expression of the constitutive active form of InR (InR CA ) in MBn decreased HAB in both sated and hungry states (Satiety:
Fig) or RNAi-mediated knockdown of AKT (S7C and S7D Fig) in α 0 β 0 MBn increased HAB.To exclude the potential developmental effects of these manipulations, we introduced the tub-Gal80 ts transgene and showed that adult-stage-specific expression of PI3K DN (S7E and S7F Fig) or knockdown of AKT (Figs 3G and S7G) in α 0 β 0 MBn increased HAB in the sated but not in the hungry state.Furthermore, AKT knockdown in α 0 β 0 MBn increased the calcium response to hot stimuli in the sated state (Fig 3H).These results suggest that Dilp2 secreted by IPCs induces PI3K/AKT signaling in α 0 β 0 MBn, which contributes to HAB and hot responses during satiety.Our results showed that constitutive expression of AKT (S7H Fig) or adultstage-specific AKT expression (Figs 3I and S7I) in α 0 β 0 MBn reduced HAB.Live brain imaging data showed that AKT overexpression in α 0 β 0 MBn decreased the hot response (Fig 3J).Taken together, our data imply that PI3K/AKT signaling inhibits HAB and hot responses by suppressing hot stimuli-induced α 0 β 0 MBn activity.Dilp6 modulates α 0 β 0 MBn activity for HAB in hungry flies via Ras/ERK signalingThe genetic silencing of dilp2 in IPCs or the inhibition of PI3K/AKT signaling in α 0 β 0 MBn increased HAB in food-sated but not in hungry flies (Figs 3C, 3G, S6B, S6C and S7A-S7G).However, manipulating InR expression in the MB affects HAB in both sated and hungry states (Fig 2), suggesting the existence of signals other than Dilp2 for HAB modulation during the hungry state.It has been shown that Dilp6 is secreted by the fat body during starvation[19], which prompted us to examine whether manipulating Dilp6 expression affects HAB.We observed that dilp6 loss-of-function mutant flies (Dilp6 LOF ) exhibited increased HAB when hungry but not when sated (Fig4A).In addition, the calcium response to the hot stimulus in α 0 β 0 MBn was significantly increased only in hungry Dilp6LOF flies (Fig 4B).Constitutive knockdown of dilp6 in the fat body via cg-GAL4 increased HAB in hungry but not in the sated state (S8A Fig).Moreover, adult-stage-specific knockdown of dilp6 in the fat body increased HAB (Figs 4C and S8B) and enhanced the hot response (Fig 4D) in α 0 β 0 MBn, specifically in hungry flies.Conversely, adult-stage-specific overexpression of dilp6 in the fat body reduced ) or a single fly in calcium imaging experiments (B, D, F, H, J).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (C, E, G, I) or the unpaired two-tailed t test ( Fig).Similarly, adultstage-specific knockdown of Ras, Raf, or Erk in α 0 β 0 MBn induced a strong HAB, specifically in the hungry state (Figs 4G and S9G-S9K).This phenotype is similar to that observed in dilp6 loss-of-function mutants (Fig 4A) and flies with dilp6 knockdown in the fat body (Fig 4C).Live calcium imaging data also support the notion that RNAi-mediated Erk knockdown in α 0 β 0 MBn increased hot response only in hungry but not in sated flies (Fig 4H).Constitutive or adult-stage-specific expression of Erk in α 0 β 0 MBn reduced HAB (Figs 4I, S9L and S9M) and the hot response of α 0 β 0 MBn (Fig 4J) in hungry flies, which supports the notion that Dilp6/ ERK signaling inhibits the hot stimulus-induced α 0 β 0 MBn activity specifically in the hungry state.

Fig 4 .
Fig 4. Dilp6 produced by the fat body regulates ERK for HAB during the hungry state.(A)dilp6 loss-of-function mutant flies (dilp6 LOF ) showed increased HAB when hungry (Satiety: P-values: 0.6176, 0.4785, 0.8634, and 0.7292 from left to right; Hunger: P-values: 0.0003, 0.0029, 0.0222, and 0.4018 from left to right).(B) α 0 β 0 MBn showed increased hot response in dilp6 LOF mutant background specifically when hungry but not when sated (P = 0.2043 for satiety; P = 0.0002 for hunger).(C) Adult-stage-specific knockdown of dilp6 in the fat body increased HAB in hungry but not in sated flies (Satiety: Pvalues: 0.7112, 0.4332, 0.748, and 0.4753 from left to right; Hunger: P-values: 0.0006, 0.0002, 0.0332, and 0.0732 from left to right).(D) Genetic knockdown of dilp6 in the fat body increased hot response of α 0 β 0 MBn specifically in the hungry state (P = 0.8682 for satiety; P = 0.0328 for hunger).(E) Adult-stage-specific expression of dilp6 in the fat body decreased HAB in the hungry state (Satiety: P-values: 0.2437, 0.2066, 0.3148, and 0.2978 from left to right; Hunger: P-values: <0.0001, <0.0001, <0.0001, and 0.0194 from left to right).(F) Genetic expression of dilp6 in the fat body decreased hot response of α 0 β 0 MBn in the hungry state (P = 0.5214 for satiety; P = 0.002 for hunger).(G) Adult-stage-specific knockdown of Erk in α 0 β 0 MBn increased HAB during hunger (Satiety: P-values: 0.9053, 0.6805, 0.4785, and 0.2593 from left to right; Hunger: P-values: <0.0001, 0.0011, 0.0026, and 0.108 from left to right).(H) Genetic knockdown of Erk in α 0 β 0 MBn increased hot responses of α 0 β 0 MBn during hunger but not during satiety (P = 0.2155 for satiety; P = 0.0361 for hunger).(I) Adult-stage-specific expression of Erk in α 0 β 0 MBn decreased HAB during the hungry state (Satiety: P-values: 0.6868, 0.7727, 0.7495, and 0.0552 from left to right; Hunger: P-values: 0.001, 0.0003, 0.0002, and 0.0121 from left to right).(J) Genetic expression of Erk in α 0 β 0 MBn decreased the hot response of α 0 β 0 MBn in the hungry state (P = 0.4394 for satiety; P = 0.0003 for hunger).The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in the behavioral assay (A, C, E, G, I) or a single fly in calcium imaging experiments (B, D, F, H, J).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (C, E, G, I) or the unpaired twotailed t test (A, B, D, F, H, J). *P < 0.05; ns, not significant.HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.
Fig 4. Dilp6 produced by the fat body regulates ERK for HAB during the hungry state.(A)dilp6 loss-of-function mutant flies (dilp6 LOF ) showed increased HAB when hungry (Satiety: P-values: 0.6176, 0.4785, 0.8634, and 0.7292 from left to right; Hunger: P-values: 0.0003, 0.0029, 0.0222, and 0.4018 from left to right).(B) α 0 β 0 MBn showed increased hot response in dilp6 LOF mutant background specifically when hungry but not when sated (P = 0.2043 for satiety; P = 0.0002 for hunger).(C) Adult-stage-specific knockdown of dilp6 in the fat body increased HAB in hungry but not in sated flies (Satiety: Pvalues: 0.7112, 0.4332, 0.748, and 0.4753 from left to right; Hunger: P-values: 0.0006, 0.0002, 0.0332, and 0.0732 from left to right).(D) Genetic knockdown of dilp6 in the fat body increased hot response of α 0 β 0 MBn specifically in the hungry state (P = 0.8682 for satiety; P = 0.0328 for hunger).(E) Adult-stage-specific expression of dilp6 in the fat body decreased HAB in the hungry state (Satiety: P-values: 0.2437, 0.2066, 0.3148, and 0.2978 from left to right; Hunger: P-values: <0.0001, <0.0001, <0.0001, and 0.0194 from left to right).(F) Genetic expression of dilp6 in the fat body decreased hot response of α 0 β 0 MBn in the hungry state (P = 0.5214 for satiety; P = 0.002 for hunger).(G) Adult-stage-specific knockdown of Erk in α 0 β 0 MBn increased HAB during hunger (Satiety: P-values: 0.9053, 0.6805, 0.4785, and 0.2593 from left to right; Hunger: P-values: <0.0001, 0.0011, 0.0026, and 0.108 from left to right).(H) Genetic knockdown of Erk in α 0 β 0 MBn increased hot responses of α 0 β 0 MBn during hunger but not during satiety (P = 0.2155 for satiety; P = 0.0361 for hunger).(I) Adult-stage-specific expression of Erk in α 0 β 0 MBn decreased HAB during the hungry state (Satiety: P-values: 0.6868, 0.7727, 0.7495, and 0.0552 from left to right; Hunger: P-values: 0.001, 0.0003, 0.0002, and 0.0121 from left to right).(J) Genetic expression of Erk in α 0 β 0 MBn decreased the hot response of α 0 β 0 MBn in the hungry state (P = 0.4394 for satiety; P = 0.0003 for hunger).The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in the behavioral assay (A, C, E, G, I) or a single fly in calcium imaging experiments (B, D, F, H, J).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (C, E, G, I) or the unpaired twotailed t test (A, B, D, F, H, J). *P < 0.05; ns, not significant.HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.

Fig 6 .
Fig 6.Cholinergic mALT neurons convey hot information to α 0 β 0 MBn.(A) The morphology of mALT neurons in the fly brain.Brain is counterstained with anti-DLG antibody.(B) The synaptic bouton-like structure (arrows) of mALT in the MB calyx region.Brain is counterstained with anti-DLG antibody.(C) The co-localization (arrows) of anti-ChAT immunostaining signals (magenta) and mALT axons (green).(D)Optogenetic silencing of mALT activity via GtACR2 inhibited HAB in both hungry and sated states (Satiety: P-values: 0.0003, <0.0001, 0.0001, and 0.0271 from left to right; Hunger: P-values: 0.0001, 0.001, 0.0103, and 0.001 from left to right).(E) Adult-stage-specific knockdown of ChAT in mALT inhibited HAB during both feeding states (Satiety: P-values: <0.0001, <0.0001, 0.0029, and <0.0001 from left to right; Hunger: P-values: <0.0001, 0.0005, <0.001, and <0.0001 from left to right).(F) Hot stimulus evoked the calcium response in hot glomerulus of mALT and this calcium response was not significantly different between sated (black) and hungry (red) flies (P = 0.7554).The GCaMP intensity changes (ΔF/F 0 ) in mALT dendrites were recorded and analyzed.(G) Optogenetic activation of mALT via CsChrimson evoked calcium responses in α 0 β 0 MBn in flies carry R35B12-lexA/CsChrimson; VT40053/lexAop-GCaMP7 (green) but not in flies carrying R35B12-lexA/CsChrimson; +/lexAop-GCaMP7 flies (black) (P < 0.0001 for satiety; P < 0.0001 for hunger).(H) The calcium responses to hot stimuli in α 0 β 0 MBn do not differ significantly between sated (black) and hungry (red) flies when InR DN transgene is expressed in α 0 β 0 MBn (P = 0.6093).(I) The calcium responses to hot stimuli in α 0 β 0 MBn do not differ significantly between sated (black) and hungry (red) dilp2 -/-and dilp6 LOF double mutant flies (P = 0.4850).The GCaMP intensity changes (ΔF/F 0 ) in MB β 0 lobe were recorded and analyzed.The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied.Each N represents either a group of 15 flies analyzed together in the behavioral assay (D, E) or a single fly in calcium imaging experiments (F-I).Data are represented as mean ± SEM with dots representing individual values.The data underlying this figure can be found in S1 Data.Data were analyzed by one-way ANOVA followed by Tukey's test (D, E) or the unpaired two-tailed t test (F-I).*P < 0.05; ns, not significant.HAB, hot avoidance behavior; mALT, medial antennal lobe tract; MBn, mushroom body neuron; SEM, standard error of mean.

Fig 8 .
Fig 8.The neuronal mechanism of feeding state-dependent hot avoidance in Drosophila.The hot stimulus is conveyed through cholinergic mALT neurons to α 0 β 0 MBn (green) and α 0 β 0 MBn activity is positively correlated with HAB.In the food-sated state, Dilp2 is secreted from IPCs to inhibit the hot response of α 0 β 0 MBn by inducing PI3K/AKT signaling that causes a weak HAB and consequently flies decrease their hot avoidance for increasing their metabolism.On the other hand, Dilp2 is no longer released from IPCs in the hungry state.Instead, Dilp6 is released from the fat body to inhibit the hot response of α 0 β 0 MBn by inducing Ras/ERK signaling.The inhibition efficiency of Ras/ERK in the hungry state is not as strong as PI3K/AKT signaling, consequently flies exhibit strong hot responses in α 0 β 0 MBn that contributes to a strong HAB, thereby increasing the hot avoidance of flies to reduce their metabolism for saving energy in the hungry state.HAB, hot avoidance behavior; IPC, insulin-producing cell; mALT, medial antennal lobe tract; MBn, mushroom body neuron; SEM, standard error of mean.